INNOVATIVE ANALYTICAL SOLUTIONS FOR THE DAIRY INDUSTRY Pierre Broutin,

1. INNOVATIVE ANALYTICAL SOLUTIONS FOR THE DAIRY INDUSTRY Pierre Broutin, Managing Director/Western Europe Senior Scientist IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

2. Bentley CombiFTS Rapid and Accurate Determination of Milk Chemical Composition and Somatic Cells content Milchprüfring Baden-Württemberg, Germany 5xCombiFTS 500, 1 IBC150 IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

3. Bentley Bartec CombiFTS 600Up to 600 samples/hour IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

4. Bentley Bartec CombiFTS 500 IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

5. Automation CombinationwithRaudszusConveyor IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

6. Bentley ILAS CombiFTS 600 A Unique Solution – up to 600 samples/hour IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

7. Bentley CombiFTS Ilas 600The optimum solution Complete and fullyautomated standardisation of yoursamplepreparation and QC: • Sampleswarmed up in a water bath, shaken and identifiedautomatically (RFID, Barcode) • Samplesuncappedautomatically • Samplesreplaced in the trayafteranalysis • Pilot samplesautomaticallyinserted in the process • Highestthroughput IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

8. Bentley CombiFTS Somacount FCM (Flow Cytometry) Bentley FTS (FTIR) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

9. Bentley FTSMilk Components Absorption wavebands Lactose (OH) Fatty Acids Fat B (CH) Protein (NH) Fat A (C=0) Frequency of infrared radiation absorbed depending upon type of molecular bonds /vibration mode IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

10. Homogenizer pressure curve IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

11. Somatic Cells Height Distribution (DNA content) Somatic Cells Width Distribution (cells size) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

12. Full Sample & environment traceability • For each samples, all the following information is recorded: • Infrared Spectrum • All environmental variables that could affect the end results: - Sample, water bath, ambient, homogenizer, deck temperatures - Optical deck Moisture level and pressure - Centerburst energy level (automatic alignment) - Homogenizer pressure - Vibrations…. • Somatic cells peak distributions IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

13. FTS Infrared Applications 1) Traditional Components → Fat, Protein, Lactose, Solids, SNF, MUN, FPD 2) New Components available: • Citric Acid • Casein • pH • FFA • Fatty Acids profile (Saturated, Unsaturated, Mono & Poly Unsaturated,C16:0, C18:0, C18:1, short, medium, long chain FA ….) • Milk Ketone bodies (Acetone/BHB) 3) New developments - Unique Solutions • Blood BHB (patent pending) • Lactoferrin • Iodine value (often used to determine the amount of unsaturation in fatty acids (C=C)) • 50 more Fatty Acids calibrations under development) … IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

14. Blood BHB Determination by infrared spectroscopyfor the monitoring of the cows metabolic activity and detection of ketosis (patent pending) • Why predicting Blood BHB instead of Milk BHB? • Milk BHB is unstableand levels very low, close to the infrared • technology detection level • Milk BHB content will decrease rapidly if the milk is not stored at • 4°C right after milking • Thus, The determination of milk BHB is not very reliable and only targets the • Milk BHB content • The development of a calibration based on the Blood BHB allows not only • to take into account the variation of the ketone bodies but also all the • variations in the milk composition that are directly or indirectly linked to • the metabolic disorder, and particularly the milk fatty acids profile IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

15. Blood BHB Determination by infrared spectroscopyfor the monitoring of the cows metabolic activity and detection of ketosis (patent pending) Blood BHB Infrared calibration on 696 cow samples IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

16. Blood BHB Determination by infrared spectroscopyfor the monitoring of cows metabolic activity and detection of ketosis (patent pending) Correlation spectrum between the Blood BHB content and the milk spectra The spectral zone with the highest correlation are related to the milk fatty acids profile IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

17. Lactoferrinfor the early detection of mastitis and monitoring of the sanitary status of the mammary gland • Lactoferrin (LF) is an iron binding antimicrobial glycoprotein present in milk • Plays a role in pre-immune host defense • Strong association between LF concentration and occurrence of mastitis • Could be used to check sanitary status of the mammary glands in the herd • Previously reported normal milk LF concentrations in lactation period were between 250-404 µg/ml. • 150 DHIA samples selected: - 50 from healthy cow (<200 kSCC) - 50 with accute mastitis () - 50 with chronical mastitis (> 800 kSCC/3-4 controls) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

18. Lactoferrin (LF)for the detection of mastitis and monitoring of the sanitary status of the mammary gland • Correlation between Log LFir and Log SCC with Log LFref significantly better than normal scale • Correlation between Log (Lfir) and Log(LFref) equivalent to correlation between Log SCC and Log LFref • LF seems to be a good indicator of Cow Mastitis Status and could especially be used to differentiate • healthy from accute or chronical mastitis • It has been suggested that bovine milk LF play an important role in host defense against Escherichia • Coli mastitis, On the other end, LF thought to have little antibacterial effect on Staphylococcus aureus • or Streptococcus agalactia IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

19. Lactoferrinfor the detection of mastitis and monitoring of mammary gland sanitary status IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

20. Quantitative vs Qualitative Analysis Limits of Quantitative Analysis : Requires to know the target components to be measured Requires to collect representative calibration samples and classical reference values Advantages of the Qualitative Analysis (spectral database): No classical reference values required No need initially to know the target components/contaminants but requires developing a spectral database of “normal/target” samples Qualitative Analysis can be used : → to detect abnormal samples → to detect adulterated samples (melamine…) → to improve calibration robustness (on-going calibration) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

21. HOW TO DEVELOP A SPECTRAL DATABASE? STEP1: SPECTRA COLLECTION OF « NORMAL /TARGET » SAMPLES ν (cm-1) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

22. DETECTION OF ABNORMAL/ADULTERATED SAMPLES STEP3: PROJECTION OF NEW/UNKNOWN SAMPLES IN SPECTRAL DATABASE ν (cm-1) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

23. DETECTION OF ABNORMAL/ADULTERATED SAMPLES Spectral Approach: -Highly sensitive and exhaustive approach across the entire FTIR spectrum = fingerprinting Spectral approach can be applied: -At the Individual Cow Level(monitoring between controls) - At the Herd Level (checking cow vs herd) - Between herds (checking herd vs herd) - On bulk tank milk samples - To check dairy plant milk process IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

24. Bentley FTSFully Optimir Compatible (www.optimir.eu) • The project aims to develop innovative farm management web applications that will use the spectral analysis of the Milk Recording samples to enable a sustainable and profitable management of the milk production • The Mid Infrared (MIR) spectrumis used as a mirror of the cows' state giving indicators on their: Fertility Feeding Health Rejection of pollutants (methane) Milk Quality FTS spectra are readily available on the instrument as well as all the environmental variables that could affect the quality of the spectra. All data can be exported in real time together with the results IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

25. Bentley FTS/DairySpec FTOn-going & Future Developments 1) New calibrations/applications - Quantitative (Fatty acids (C14:0, C4:0, C6:0, C8:0, C10:0, C12:0, C18:3, C18:3n-3) , FFA, Ketones bodies….) - Qualitative (fingerprinting) - Blood BHB, TEM, Lactoferrin, Iodine value…. 2) Rapid determination of milk adulteration - by contaminants - by milk from different animal species 3) Detection of abnormal samples: - Outside the calibration range - Outside laboratory «normal » spectral database 4) Use of 3rd party calibration/models (Phenofinlait/Optimir…) 5) Somatic cells differentiation IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

26. Somatic Cells Differentiation • Mastitis in dairy cattle is the a persistent inflammatory reaction of the udder tissue. This mammary gland infection is the most common disease in dairy cattle. It is also the most costly to the dairy industry (loss of milk production, culling, treatments, inhibitors…) • Milk from cows with mastitis has a higher somatic cells content. The total Somatic Cells Count not always sufficient to identify the origin of the infection and reduce the number the incidence of mastitis in the herd. • New tools have been developed to improve the mastitis diagnostic or to anticipate the onset of mastitis: • Use of the Pathoproof PCR test to identify and quantify the 16 most predominant mastitis bacteria on individual cow or on bulk milk • Use of the Lactoferrin which plays a role in the pre-immune host defense system to detect on the onset of mastitis (prevention) • Use of BactoCount to screen DHIA Milk Samples • Somatic Cells differentiation (PMN, Macrophages (M), Lymphocytes…) • Chronic Mastitis: High SCC, High Macrophages, Low PMN • Acute Mastitis: Higher SCC, High PMN (up to 90% of SCC) IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

27. Somatic Cells Differentiation IBC vs SCC on Individual Cow Milk Samples IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

28. Somatic Cells Differentiation IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

29. CombiFTS Main Features & Benefits IDF PRIVILEGED SUPPORTING PARTNER • Full FT-MIR High Resolution Spectrum (8 cm-1) • Up to 600 samples/hour • Fat/Protein/Lactose/Solids/MUN/FPD/Fatty Acids profile+++ • Fully compliant with ICAR/IDF/ISO requirements • Automatic performance checks and adjustment • Automatic rinse/auto-zero function (programmable) • Completely open system+full potential of an FTIR deck (spectra access/calibration development) • Permanent database storage of all raw data (spectra) and diagnostics (complete traceability) • Spectral Database retroactive valorisation with new calibration models • Extensive network remote control and data access Full potential of an FTIR deck – no limitations IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin

30. Thankyou for your attention ! pbroutin@bentleyinstruments.com www.bentleyinstruments.com IDF/ICAR Congress – Manufacturer Showcase May 16, 2014 Berlin